Functional identification of unknown proteins discovered in sequence and structural genome projects is a major challenge for postgenomic biology. This Program Project develops an integrated sequence-structure based strategy for functional assignment of unknown proteins by predicting the substrate specificities of unknown members of the functionally diverse enolase and amidohydrolase superfamilies that share the ubiquitous (beta/alpha)8-fold. The unimolecular reactions catalyzed by members of each of these superfamilies share common mechanistic strategies, so the success of specificity predictions can be readily evaluated. This project integrates expertise in functional enzymology (protein purification, measurement of function, and determination of mechanism), structural enzymology (high resolution x-ray structural analysis), and, for the first time, computational biology (bioinformatics, homology modeling, and molecular docking). These diverse, but complementary, approaches for obtaining clues regarding the identity of the substrate will efficiently restrict the identities of the substrates for unknown members of these superfamilies. The integrated strategy developed in this Project will be generally applicable to deciphering the ligand specificities of any functionally unknown protein. The goals of this project extend the contribution of the Protein Structure Initiative funded by NIGMS that seeks to obtain structures for proteins of unknown function that will allow reliable homology modeling.